Cardiovascular disease (CVD) takes many forms. Heart attacks are only one form of cardiovascular disease. Others include hypertension (high blood pressure), coronary artery disease, rheumatic heart disease, stroke, and peripheral vascular disease with gangrene. According to the American Heart Association, CVD has been the No. 1 killer in the United States every year since 1900 but 1918. Nearly 2,600 Americans die of CVD each day, an average of 1 death every 34 seconds. CVD claims more lives each year than the next 5 leading causes of death combined, which are cancer, chronic lower respiratory diseases, accidents, diabetes mellitus, and influenza and pneumonia. American Heart Association, Heart Disease and Stroke Statistics—2004 Update, page 3 (http://www.americanheart.org).
In 2004, the estimated direct and indirect cost of CVD is $368.4 billion. CVD does not only occur in adults. Atherosclerosis and other heart diseases, such as stroke, are serious and largely unrecognized problems that affect thousands of children.
Platelets, which are small cell derived bodies that normally circulate in the bloodstream, help the body defend itself against bleeding and blood loss. They function by sticking to the vessel surface and aggregating (sticking together) and helping in the formation of a clot at the site of bleeding. The process of creating a blood clot, which is called thrombosis, is beneficial to a person with severe bleeding. However, blood clots can also cause severe problems, particularly when they form in the vessels of the heart or brain.
Under normal healthy conditions, the different cellular components of flowing blood (such as red and white blood cells and platelets) are unable to stick to the inner lining of blood vessels and cause a blockage that disrupts blood flow. However, when there is injury to the inner lining of blood vessels, such as caused by injurious pulsatile flow, coronary artery stent placement, buildup of fatty deposits, or connective tissue and cellular proliferation (atherosclerosis) within blood vessels, the lining of the vessels (the endothelium) is less resistant to the formation of dangerous blood clots. As a result of the injury, platelets in the circulating blood begin to accumulate, along with isolated monocytes and macrophage/foam cells (lipid filled macrophages), and begin to accumulate excess cholesterol, fats, and connective tissue (collagen and aelastin). Cholesterol, fat and other inflammatory substances (cytokines and chemokines) are also attracted to minor injuries in arterial walls that arise from high blood pressure.
Plasma proteins such as fibrin and fibrinogen also accumulate in atheromata. Meanwhile, the circulating blood and tiny blood vessels in the artery walls (vasa vasorum) continue to supply more fat, cholesterol, connective tissue and cells to fibrous lesions so that the deposits continue to grow. In humans, by the mid-thirties and early forties, atherosclerotic deposits are seen throughout the vascular tree and can become calcified, as chalky minerals accumulate that fill in the fibrous scar tissue. Most young adults have atherosclerotic plaque not only in the heart vessels, but also along the entire length of the ascending aorta, leading toward the brain, and along the iliac and femoral arteries nourishing the organs in the pelvic region. These complicated lesions set the stage for stroke, heart attack or peripheral vascular disease. In addition to narrowing the arteries, atherosclerotic plaque may ulcerate and form thrombi, which are made up chiefly of coagulated blood platelets.
A variety of stimuli, including high blood pressure, high blood sugar (diabetes), acute inflammation, poisonous chemicals like tobacco components, and even stress may make atherosclerotic fat deposits (plaques) unstable. This is particularly true when plaques are rich in fat (cholesterol) and white blood cells (inflammatory cells). An unstable plaque may crack or rupture and expose its contents to flowing blood. In its own defense, the body attempts to heal this injury by forming a blood clot over the damaged area. The formation of a blood clot occurs in several steps. First, platelets adhere to the ruptured plaque surface. They then begin to stick together in a process known as aggregation. The growing platelet aggregate forms a surface on which the process of coagulation can occur: through a series of enzymatic reactions involving serine proteases, a net-like substance (fibrin) forms, linking platelets together, red blood cells are trapped within the fibrin meshwork, and a blood clot results.
Blood clots may form when blood circulation is slowed, or they may develop around atheromata and cause an active, acute obstruction of the arteries. An embolus, or detached thrombus, can also drift downstream to smaller-diameter blood vessels where it may eventually become lodged like a boulder in a stream. When this happens, blood supply may be completely shut off, producing an infarction, or localized death, of a segment of the brain, the heart muscle, the lungs, the legs or the feet. Other complications may also result from the buildup of atherosclerotic plaque. When tissue in the wall of an artery under an atheroma bleeds, hemorrhaging may result. An abscess, or localized infection, may also develop beneath the hardened deposit, leading to injury and disease.
Blood clots can be especially dangerous for patients with heart disease. A heart attack (myocardial infarction) results when a blood clot interrupts or blocks blood flow to the heart, which starves the heart muscle of oxygen and causes heart muscle cells to die; the same process in the brain causes a stroke (cerebral infarction).
Vascular cells become multi-functional at the interface of thrombosis and inflammation with accumulating evidence linking inflammatory responses to pro-thrombotic stimuli. Endothelial cells provide both a barrier and link between activation of coagulation and innate immune pathways. Inflammation shifts the endothelial cell balance from an anti-thrombotic state to a pro-coagulant state, with accompanying conversion of pro-enzymes to active serine proteinases in the clotting cascade. Activated serine proteinases promote adhesion and activation of platelets on the endothelial cell surface through the expression of adhesion molecules. Adhesion molecules expressed on the endothelial cells, as part of a pro-coagulant response, recognize, bind and decelerate leukocyte motion in the blood stream and support emigration of leukocytes into the vessel wall. The transmigrated leukocytes are activated and express inflammatory molecules that promote plaque development. Activated leukocytes also promote the production and activation of matrix metalloproteinases (MMPs), which degrade extracellular matrix proteins and results in plaque instability and rupture. Unstable ruptured plaque exposes of the underlying necrotic contents to the circulating blood, causing arterial occlusion (heart attacks and strokes) and progression of plaque growth, atherothrombosis.
Exposure of the inner plaque core after rupture or erosion introduces the plaque collagen and lipids to the cells in the circulating blood and also exposes tissue factor a key mediator of the extrinsic clotting cascade and a known linking protease for clot formation and inflammatory responses. With exposure of the highly thrombotic plaque core contents, platelets, monocytes and T cells are activated resulting in further platelet activation, fibrin deposition and activation of monocytes/macrophages with a continuing cascaded of actions leading to vessel occlusions.
Thrombotic and inflammatory responses in cells are accompanied by surface receptor reorganization and structural alterations in cellular membranes, which are reflected in microviscosity (the reciprocal of membrane fluidity) changes of the amphipathic lipid bilayer. Altered membrane fluidity has been detected in many disease states, e.g, thrombocythaemia, hyperlipidimia, hypercholesterolaemia, hypertension, diabetes mellitus, obesity, sepsis, and acute coronary syndromes are among others. (Zalai et al., J Am Coll Cardiol 2001, 38:1340-47). Optimal conformation of membrane-associated receptors protects membrane's function and is maintained by specific microviscosity conditions. Accordingly, membrane fluidity acts as a marker in defining the activation state of a cell.
Serine proteinase inhibitors (“serpins”) make up a superfamily of related proteins and have been found encoded by poxviruses from four different genera. The myxoma viral secreted serine proteinase inhibitor, SERP-1, inhibits the thrombolytic proteases urokinase- and tissue-type plasminogen activators (uPA and tPA respectively) and plasmin in vitro and has demonstrated profound anti-inflammatory activity in a wide range of animal models. (U.S. Pat. No. 5,686,409; U.S. Pat. No. 5,917,014; U.S. Pat. No. 5,939,525; Nash et al., J. Biol. Chem. 1998, 273: 20982-91; Lomas et al., J. Biol. Chem. 1993, 268: 516-21; Lucas et al., Circulation 1996, 94: 2694-2705; Lucas et al., J Heart Lung Transplant 2000, 19:1029-1038; Miller et al, Circulation 2000, 101:1598-1605; Bot et al., Circ. Res. 2003, 93: 464-471; Brahn et al., American College of Rheumatology Meeting; Nov. 13-17, 1999; Boston; Bedard et al., Abstract 1143, American Society of Transplant Surgeons 2002; Wang et al., Abstract, 7th International Congress of Xenotransplantation 2003; Nash et al., J Biol Chem 1998, 273(33):20982-91; Dai et al., J of Biol Chem 2003, 278(20): 18563-72).
Since platelets play a key role in the body's response to injury in an artery and in beginning the process of forming a blood clot, interrupting that process has become an important part of the battle against heart disease and stroke. Antiplatelet therapy drugs can interfere with platelet function and are classified into 3 categories: Those that prevent cardiovascular diseases (primary prevention), those that treat an acute disease, and those that treat a chronic disease (secondary prevention). There are both oral and intravenous drugs that inhibit platelet function and are used to treat patients with cardiovascular and cerebrovascular diseases. Three types of antiplatelet agents have been used as medical products: aspirin, ADP receptor antagonists (clopidogrel and ticlopidine) and glycoprotein IIb/IIIa inhibitors (abciximab, integrilin and tirofiban). Their effectiveness, however, is widely variable and there remains a significant clinical unmet need. These agents while preventing acute thrombosis in native atherosclerotic disease or after intervention (angioplasty and stent implant) have limited anti-inflammatory activity. The ADP receptor antagonists and glycoprotein IIb/IIIa antagonists do not prevent atherosclerosis nor restenosis after angioplasty or stent implant. Aspirin and plavix reduce thrombtic occlusion in unstable plaque leading to stroke and myocardial infarctions but many patients are still admitted with unstable coronary and acrotid syndromes with associated progression to MI, CVA or PVO (peripheral vascular occlusion suggesting that the anti-inflammatory actions of ASA are only partially effective in preventing disease progression. This is especially true in the aforementioned diseases where available therapy is either of limited effectiveness/route of administration or is accompanied by unwanted side effect profiles. Thus, there is a need for a safe, effective clinical agent for antiplatelet/antithrombosis therapy.
It has been discovered in accordance with the present invention that SERP-1 displays anti-thrombotic and antiplatelet activities in mammals.